1. Field of the Invention
The present invention relates to a method for measuring a concentration, of a substrate in a sample liquid by allowing the substrate to react with an enzyme that can specifically react with the substrate and then quantifying, after a predetermined period of time, the change in the concentration of a material that has been changed through the reaction. More particularly, the present invention relates to an accurate, rapid and easy method for measuring concentrations of a plurality of substrates, such as sucrose and glucose, contained in a sample liquid, such as fruit juice, blood, lymph and urine, by utilizing specific catalyses of enzymes in one biosensor.
2. Description of the Related Art
Various types of biosensors utilizing specific catalyses of enzymes have been recently developed. Such biosensors are advantageous in measuring a concentration of a substrate in various sample liquids, such as fruit juice, blood., lymph and urine, because they have sufficient specificity to each substrate in order to attain an accurate measurement and because they can achieve relatively easy and rapid quantification of the substrate.
Japanese Laid-Open Patent Publication No. 3-202764 and U.S. Pat. No. 5,192,415 disclose the following method for measuring a concentration of a substrate in a sample liquid by using a biosensor:
The biosensor has an insulating substrate, an electrode system including a working electrode and a counter electrode formed on the substrate, and a reaction layer including a hydrophilic polymer, an enzyme and an electron acceptor formed on the electrode system. When a sample liquid containing a substrate to be quantified is supplied to the biosensor, the enzyme and the electron acceptor in the reaction layer react with the sample liquid. The change in the concentration of a material through this reaction is electrochemically detected by the electrode system. Thus, the concentration of the substrate in the sample liquid is measured. PA1 (a) determining a relationship between a concentration of the first substrate and a response current value measured with an auxiliary testing biosensor having the same structure as the biosensor, the response current value being measured after a predetermined period of time T.sub.1, T.sub.1 beginning upon supplying a solution including the first substrate to the auxiliary testing biosensor; PA1 (b) determining a relationship between the concentration of the first substrate and a response current value measured with the auxiliary testing biosensor after a predetermined period of time T.sub.2, T.sub.2 beginning upon supplying the solution including the first substrate; PA1 (c) determining a relationship between a concentration of the second substrate and a response current value measured with another auxiliary testing biosensor having the same structure as the biosensor, the response current value being measured after the predetermined period of time T.sub.2, T.sub.2 beginning upon supplying a solution including the second substrate to the auxiliary testing biosensor; PA1 (d) supplying a sample liquid including the first and second substrates to the biosensor and applying a voltage between the working electrode and the counter electrode in the electrode system after the predetermined period of time T.sub.1, T.sub.1 beginning upon supplying the sample liquid, for measuring a response current value I.sub.1 ; PA1 (e) applying a voltage between the working electrode and the counter electrode in the electrode system after the predetermined period of time T.sub.2, T.sub.2 beginning upon supplying the sample liquid, for measuring a response current value I.sub.2 ; PA1 (f) obtaining a concentration C.sub.1 of the first substrate in the sample liquid corresponding to the current value I.sub.1 based on the relationship between the concentration of the first substrate and the response current value determined in step (a); PA1 (g) obtaining a response current value I.sub.1 ' corresponding to the concentration C.sub.1 of the first substrate in the sample liquid based on the relationship between the concentration of the first substrate and the response current value determined in step (b); and PA1 (h) obtaining a concentration C.sub.2 of the second substrate in the sample liquid corresponding to a current value calculated by subtracting the current value I.sub.1 ' from the current value I.sub.2 based on the relationship between the concentration of the second substrate and the response current value determined in step (c). PA1 supplying a plurality of solutions including the first or the second substrate respectively at known concentrations to a plurality of the auxiliary testing biosensors; PA1 applying a voltage between the counter electrode and the working electrode of each auxiliary testing biosensor after the predetermined periods of time T.sub.1 and T.sub.2, T.sub.1 and T.sub.2 beginning upon supplying each solution to each of the auxiliary testing biosensors; and PA1 measuring current values of a predetermined period of time after applying the voltage. PA1 (a) obtaining a response curve a indicating a relationship between a concentration of the first substrate and a response current value measured with an auxiliary testing biosensor after a predetermined period of time T.sub.1, T.sub.1 beginning upon supplying a solution including the first substrate to the auxiliary testing biosensor, the predetermined period of time T.sub.1 being a time required for at least part of the first substrate to react with the first enzyme; PA1 (b) obtaining a response curve b indicating a relationship between the concentration of the first substrate and a response current value measured with the auxiliary testing biosensor after a predetermined period of time T.sub.2, T.sub.2 beginning upon supplying the solution including the first substrate to the auxiliary testing biosensor, the predetermined period of time T.sub.1 being a time required for at least part of the second substrate to react with the second enzyme; PA1 (c) obtaining a response curve d indicating a relationship between a concentration of the second substrate and a response current value measured with the auxiliary testing biosensor after the predetermined period of time T.sub.2, T.sub.2 beginning upon supplying the solution including the second enzyme; PA1 (d) applying a voltage between the working electrode and the counter electrode in the electrode system after the predetermined period of time T.sub.1, T.sub.1 beginning upon supplying the sample liquid including the first substrate and the second substrate to the biosensor, to measure a response current value I.sub.1 ; PA1 (e) applying a voltage between the working electrode and the counter electrode in the electrode system after the predetermined period of time T.sub.2, T.sub.2 beginning upon supplying the sample liquid including the first substrate and the second substrate to the biosensor, to measure a response current value I.sub.2 ; PA1 (f) obtaining a concentration of the first substrate in the sample liquid corresponding to the current value I.sub.1 based on the response curve a obtained in step (a); PA1 (g) obtaining a response current value I.sub.1 ' corresponding to the concentration C.sub.1 of the first substrate in the sample liquid based on the response curve b obtained in step (b); and PA1 (h) obtaining a concentration C.sub.2 of the second substrate in the sample liquid corresponding to the current value calculated by subtracting the current value I.sub.1 ' from the current value I.sub.2 based on the response curve d obtained in step (c).
A glucose sensor will now be described as an example of this type of biosensor.
In the glucose sensor, glucose oxidase is used as an enzyme in the reaction layer. When a sample liquid containing glucose is supplied to the glucose sensor, the reaction layer is dissolved in the sample liquid. The glucose in the sample liquid is oxidized by glucose oxidase in the reaction layer. At the same time, the electron acceptor in the reaction layer is reduced. After the oxidation reaction of glucose in the sample liquid is completed, a fixed voltage is applied between the working electrode and the counter electrode in the electrode system, thereby oxidizing the reduced electron acceptor. By measuring an oxidation current caused by the application of the voltage is measured to quantify the concentration of the glucose in the sample liquid.
However, this conventional quantification method can be applied only when one kind of substrate in a sample liquid is desired to be quantified. It is impossible to measure concentrations of a plurality of substrates in a sample liquid at one time.
When a plurality of substrates in a sample liquid are to be quantified, it is necessary to use a plurality of biosensors respectively using different enzymes that can specifically react with the respective substrates. Alternatively, it is necessary to use a biosensor having a plurality of electrode systems and reaction layers containing different enzymes that can specifically react with the respective substrates. The present inventors filed U.S. patent application Ser. No. 07/961,528 (filed on Oct. 15, 1992) disclosing the latter type of biosensor. In this manner, usage of a plurality of biosensors or complication of the structure of a biosensor can not be avoided in order to quantify a plurality of substrates in a sample liquid.